The AIDS epidemic has experienced a quick growth in the past 25 years (estimated number of patients at the end of 2004: 39.4 million). The use of highly-active anti-retroviral therapies (HAART) has significantly increased the life expectancy of persons infected by HIV. However, long-term treatments have caused the selection of HAART-resistant virus variants. This situation has made it necessary to develop new compounds that may be effective in fighting said variants. The addition of the fusion inhibitor “enfuvirtide” (also known as Fuzeon, T-20 or DP178) to the arsenal of anti-HIV compounds has meant a great advance in the control of multi-resistant viruses (1-3).
This compound acts on a key process of the infective cycle: the fusion of viral and cellular membranes promoted by viral coat glycoprotein gp120/41 (4). Its active principle is the T-20 peptide, which imitates a helicoidal sequence of gp41 that wraps in the hydrophobic grooves formed in the contact area between the helices of an imperfect trimeric coiled-coil. Wrapping is required in order to close a fork that clamps the membranes and causes the fusion thereof. T-20 competitively inhibits the closing process of the fork and, therefore, inhibits the fusion (FIG. 2).
However, the high production cost of “enfuvirtide” and the appearance of virus variants that are resistant to this compound have called into question its generalised use in anti-HIV therapy. Therefore, it is necessary to work in the development of new alternative compounds which are effective fusion inhibitors, have a lower cost and may be included within integrated strategies to control the disease. If these compounds act through a mechanism different from “enfuvirtide”'s, they could be used concertedly in the elimination of viruses which have developed resistance to HAART treatments. Moreover, they could represent an alternative to standard fusion inhibition therapies in cases of resistance to “enfuvirtide”.
The amino-terminal sequence of fusogenic protein gp41 of HIV-1 is hydrophobic and highly conserved amongst the different strains, variants and clinical isolates (5). This is due to the fact that it is required in order for the protein to perform the fusogenic function (reviewed in 6). In accordance with its function, this sequence is called “fusion peptide” (hereinafter, FP). Its high degree of conservation and its functionality make this sequence a suitable therapeutic target for the development of anti-gp41 inhibitors. Previous data indicate that certain oligopeptides are capable of interfering with the fusogenic activity of gp41 (7), as well as with the interaction of synthetic FP with liposomes (8).
Patent application WO 03/104262 A2 discloses peptides designed to inhibit the fusion of protein gp41 of HIV with target cells, and patent application WO 2004/047730 A2 discloses a chemical compound with molecular weight between 200 and 1,200 Daltons, and logP between −2.0 to +5.5, which is capable of reciprocally acting with the hydrophobic cavity and blocking fusion formulation with the coiled coil of gp41.
But all these methods designed to prevent HIV infection and cure AIDS are still limited. Consequently, there is still the need to develop new viral inhibitors, specially inhibitors that are non-toxic or have an acceptably low toxicity.
There are various peptide synthesis methods that are distinguished by the physical state of the phase wherein said synthesis takes place, that is, in the liquid phase or the solid phase (see, for example, patent applications WO 2005/063791 A2, WO 2005/063792 A2 and WO 2005/063793 A2). In the solid phase, a first amino acid or peptide is bound to an insoluble support, such as a resin. Successive groups of amino acids or peptides are added until the desired peptide is obtained. Subsequently, said peptide is separated from the resin, isolated and identified.
But this invention relates to the synthesis and isolation of oligopeptides, not hydrolysable by cellular proteases, which interfere with FP, as potential new “fusion inhibitor” agents for HIV, and operate according to a mechanism that is substantially different from that of “enfuvirtide”. The oligopeptides of this invention act by blocking the insertion of the FP in the target cell membrane and interfering with the self-assembly processes that take place on the surface thereof. On the contrary, “enfuvirtide” acts in a subsequent step, by preventing closing of the fork (see FIG. 2) once the virus and the host cell have come into contact.